新疆籽用西瓜ISSR反应体系的建立及其遗传多样性研究
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摘要
【目的】研究籽用西瓜遗传多样性,对籽用西瓜品种进行遗传多样性和聚类分析,为杂种选育提供一定的理论依据。【方法】以60份籽用西瓜种质资源为材料,对ISSR引物UBC801～UBC900进行筛选。【结果】筛选出多态性较好的ISSR引物10条。利用10条ISSR引物扩增60份籽用西瓜种质资源,共获得44个等位变异,每对引物检测到的等位变异数的变幅为3～7个,平均等位变异数4. 4个。其中多态性条带为24条,10条ISSR引物的多态性信息含量(PIC)的变化范围为0. 042 3～0. 674 0,平均为0. 358 2,遗传相似系数的变异范围为0. 318～0. 992,平均为0. 779。利用UPGMA法进行聚类分析,新疆籽用西瓜分为6大组,第一组为材料47(40036),第二组为材料8(新籽瓜8号),第三组为材料10(红秀2号)、11(普通红大片)、12(新籽瓜4号)、13(紫荆红)、14(40002)、15(40004)、16(40008),第四组为材料7(新籽瓜1号),第五组为材料52(40045),其余的都归类为第六组。【结论】ISSR对籽用西瓜种质资源遗传多样性和亲缘关系确定相比其他分子标记更有优势,更为全面的基因组DNA信息,品种间较丰富的遗传多样性信息。利用ISSR标记分析60份籽用西瓜遗传多样性,参试材料来源、籽色、大小不同,亲缘关系差异较大,新疆籽用西瓜种质资源遗传多样性丰富。
【Objective】In order to explore the genetic diversity of seed-watermelons,this project aims to carry out the genetic diversity and cluster analysis of various varieties.【Method】Sixty seed-watermelon germplasm resources were employed as researching materials for screening the suitable ISSR primer pairs from UBC801 to UBC900.【Result】Ten ISSR primer pairs were chosen as the suitable primers for producing 44 allelic variation via amplification of 60 seed-watermelon germplasm resources. Moreover,each primer pair could produce 3 to 7 allelic variation and the average allelic variation number was 4. 4. Furthermore,24 polymorphism bands were generated in this project and the polymorphism information content( PIC) from ten ISSR primer pairs ranged from 0. 042,3 to 0. 674,0,and the average PIC was 0. 358,2. The genetic similarity coefficient of 60 germplasm resources varied from 0. 409 to 0. 977 and the average coefficient was 0. 779.【Conclusion】ISSR is superior to other molecular markers in determining genetic diversity and genetic relationship of seed watermelon germplasm resources compared to other molecular markers. Meanwhile,more comprehensive genomicDNA information can be seen,which can reflect the abundant genetic diversity information among different varieties. All these provide some theoretical basis for hybrid breeding.
【Objective】In order to explore the genetic diversity of seed-watermelons,this project aims to carry out the genetic diversity and cluster analysis of various varieties.【Method】Sixty seed-watermelon germplasm resources were employed as researching materials for screening the suitable ISSR primer pairs from UBC801 to UBC900.【Result】Ten ISSR primer pairs were chosen as the suitable primers for producing 44 allelic variation via amplification of 60 seed-watermelon germplasm resources. Moreover,each primer pair could produce 3 to 7 allelic variation and the average allelic variation number was 4. 4. Furthermore,24 polymorphism bands were generated in this project and the polymorphism information content( PIC) from ten ISSR primer pairs ranged from 0. 042,3 to 0. 674,0,and the average PIC was 0. 358,2. The genetic similarity coefficient of 60 germplasm resources varied from 0. 409 to 0. 977 and the average coefficient was 0. 779.【Conclusion】ISSR is superior to other molecular markers in determining genetic diversity and genetic relationship of seed watermelon germplasm resources compared to other molecular markers. Meanwhile,more comprehensive genomicDNA information can be seen,which can reflect the abundant genetic diversity information among different varieties. All these provide some theoretical basis for hybrid breeding.
引文
[1]王翠平.籽瓜抗氧化及抗肿瘤活性研究[J].安徽农业科学,2011,39(29):17 833-17 836.WANG Cui-ping.(2011). Studies on Anticancer Activities of Seeding Watermelon[J]. Journal of Anhui Agricultural Sciences,39(29):17,833-17,836.(in Chinese)
[2]林明,李承业,潘竟海,等.种植密度对籽瓜生长发育及产量的影响[J].新疆农业科学,2012,49(3):448-453.LIN Ming,LI Cheng-ye,PAN Jing-hai,et al.(2012).Effect of planting density on the growth and yield of seed-watermelon-SWBB-01[J]. Xinjiang Agricultural Sciences,49(3):448-453.(in Chinese)
[3]赵多勇,李应彪,翟金兰.籽瓜系列产品的开发现状与存在问题[J].农产食品科技,2008,2(1):45-47.ZHAO Duo-yong,LI Ying-biao,ZHAI Jin-lan,et al.(2013). Present Situation and Existing Problems in the Development of Watermelon Seed Products[J]. Agricultural Food Products Science and Technology,2(1):45-47.(in Chinese)
[4]羊杏平,刘广,侯喜林,等.利用RAPD和ISSR 2种分子标记鉴定西瓜杂交种的遗传纯度[J].江苏农业学报,2010,26(6):1 313-1 318.YANG Xing-ping,LIU Guang,HOU Xi-lin,et al.(2010).Evaluation of Genetic Purity of Watermelon Hybrid(Citrullus lanatus)Using PAPD and ISSR Molecular Markers[J]. Jiangsu Journal of Agricultural Sciences,26(6):1,313-1,318.(in Chinese)
[5]王吉明,李楠楠,尚建立,等.西瓜抗枯萎病种质资源的分子标记筛选研究[J].安徽农业科学,2015,43(32):200-202.WANG Ji-ming,LI Nan-nan,SHANG Jian-li,et al.(2015). Screening on Molecular Markers Lingked with Fusarium WiltResistance in Watermelon(Citrulls lanatus L.)Germplasam[J]. Journal of Anhui Agricultural Sciences,43(32):200-202.(in Chinese)
[6]汤谧,任俭,杜念华,等.分子标记技术在西瓜和甜瓜育种中的应用进展[J].贵州农业科学,2013,41(4):19-22.TANG Mi,REN Jian,DU Nian-hua,et al.(2013). Research Progress in Molecular Maker Technology Application in Watermelon and Melon Breeding[J]. Guizhou Agricultural Sciences,41(4):19-22.(in Chinese)
[7]张屹,刘建雄,梁志怀,等.不同西瓜品种枯萎病抗性鉴定方法比较[J].长江蔬菜,2015,(12):50-52.ZHANG Yi, LIU Jian-xiong, LIANG Zhi-huai, et al.(2015). Comparison of Resistance Identification Methods for Different Watermelon Cultivars[J]. Journal of Changjiang Vegetables,(12):50-52.(in Chinese)
[8]戴照义,王运强,刘志雄,等.西瓜种质资源的遗传多样性分析[J].中国瓜菜,2015,28(6):5-9.DAI Zhao-yi,WANG Yun-qiang,LIU Zhi-xiong,et al.(2015). Analysis on genetic diversity of watermelon germplasm resources[J]. China Cucurbits and Vegetables,28(6):5-9.(in Chinese)
[9]石磊,王萍,杨静,等.籽用西瓜种质资源SSR分析及初级核心种质库构建[J].西北植物学报,2016,36(6):1 125-1 134.SHI Lei,WANG Ping,YANG Jing,et al.(2016). Genetic Diversity Analysis and Construction of Seed Watermelon Core Germplasm Resources by SSR[J]. Acta Botanica Boreali-Occidentalia Sinica,36(6):1,125-1,134.(in Chinese)
[10]张楠,栾非时,高鹏. 4份野生瓜类材料抗病性及形态特征和SSR亲缘关系研究[J].园艺学报,2012,39(5):905-914.ZHANG Nan,LUAN Fei-shi,GAO Peng.(2012). The Study of Disease Resistant,Morphological Characteristics and Genetic Relationship Using SSR Markers in 4 Materials of Wild Cucurbit[J]. Acta Horticulturae Sinica,39(5):905-914.(in Chinese)
[11]Zietkiewicz,E.,Rafalski,A.,&Labuda,D.(1994). Genome fingerprinting by simple sequence repeat(ssr)-anchored polymerase chain reaction amplification. Genomics,20(2):176-183.
[12]李严,张春庆.西瓜杂交种遗传多态性的SRAP标记分析[J].园艺学报,2005,32(4):643-647.LI Yan,ZHANG Chun-qing.(2005). Studies on Genetic Diversity with a Molecular Marker SRAP of Watermelon Hybrids[J]. Acta Horticulturae Sinica,32(4):643-647.(in Chinese)
[13]李晓慧,田朝阳,王从彦. SRAP分子标记分析西瓜遗传多态性[J].生物技术,2007,17(3):23-26.LI Xiao-hui, TIAN Chao-yang, WANG Cong-yan.(2007). Genetic Polymorphism Analysis by Sequence-related Amplified Polymorphism Markers in Citrullus lanatus[J]. Biotechnology,17(3):23-26.(in Chinese)
[14]李朋飞,霍秀爱,程永强,等.基于SRAP的西瓜种质资源遗传多样性评价[J].中国农业科技导报,2013,15(2):89-96.LI Peng-fei,HUO Xiu-ai,CHENG Yong-qiang,et al.(2013). Assessment of Genetic Diversity in Watermelon Based on SRAP Analysis[J]. Journal of Agricultural Science and Technology,15(2):89-96.(in Chinese)
[15]易克,徐向利,卢向阳,等.利用SSR和ISSR标记技术构建西瓜分子遗传图谱[J].湖南农业大学学报(自然科学版),2003,29(4):333-337.YI Ke,XU Xiang Li,LU Xiang-yang,et al.(2003). Construction of Molecular Genetic Map of Watermelon by SSR and ISSR Technology[J]. Journal of Hunan Agricultural University(Natural Science),29(4):333-337.(in Chinese)
[16]羊杏平,刘广,侯喜林,等.利用RAPD和ISSR分子标记对西瓜作图亲本的多态性分析[J].江苏农业学报,2009,25(5):1 100-1 107.YANG Xing-ping,LIU Guang,HOU Xi-lin,et al.(2009).Polymorphism Analyses of Watermelon(Citrullus lanatus)Mapping Parents Using RAPD and ISSR Molecular Markers[J].Jiangsu Journal of Agricultural Science,25(5):1,100-1,107.(in Chinese)
[17]刘娟,廖康,赵世荣,等.利用ISSR分子标记构建新疆野杏核心种质资源[J].中国农业科学,2015,48(10):2 017-2 028.LIU Juan,LIAO Kang,ZHAO Shi-rong,et al.(2015). The Core Collection Construction of Xinjiang Wild Apricot Based on ISSR Molecular Markers[J]. Scientia Agricultura Sinica,48(10):2,017-2,028.(in Chinese)
[18]柳唐镜,于海彬,张棵,等.红籽瓜种质资源亲缘关系RAPD分析[J].中国农学通报,2010,26(22):264-270.LIU Tang-jing,YU Hai-bing,ZHANG Ke,et al.(2010).Genetic Relationships Analysis of Red-seed Edible Seed Watermelon Germplasm Resources by Using RAPD Markers[J]. Chinese Agricultural Science Bulletin,26(22):264-270.(in Chinese)
[19]刘海河,侯喜林,张彦萍.西瓜ISSR-PCR体系的正交优化研究[J].果树学报,2004,21(6):615-617.LIU Hai-he,HOU Xi-lin,ZHANF Yan-ping.(2004).Optimization of ISSR-PCR System in Watermelon[J]. Journal of Fruit Science,21(6):615-617.(in Chinese)
[20]黄歆贤. ISSR和SSR标记在大豆、油菜、西瓜种子真实性和纯度鉴定中的比较[D].杭州:浙江大学硕士学位论文,2011.HUANG Xin-xian.(2011). Comparison of ISSR and SSR markers for seed genuineness and variety purity identification in soybean,rape and watermelon[D]. Master Dissertation. Zhejiang University,Hangzhou.(in Chinese)
[21]杨生保,杨涛,李宁,等.番茄ILs果实性状的主成分分析与聚类分析[J].植物遗传资源学报,2015,16(4):907-913.YANG Sheng-bao,YANG Tao,LI Ning,et al.(2015). Principal Component and Cluster Analysis Based on Fruit Traits in ILs of Tomato[J]. Journal of Plant Genetic Resources,16(4):907-913.(in Chinese)
[22]杨生保,李宁,杨涛,等.基于SSR标记的制干辣椒品种资源遗传多样性分析[J].分子植物育种,2015,13(4):840-845.YANG Sheng-bao,LI Ning,YANG Tao,et al.(2015). Genetic Diversity of Dried-Orientation Pepper Cultivars Revealed by SSR Marker[J]. Molecular Plant Breeding,13(4):840-845.(in Chinese)
[2]林明,李承业,潘竟海,等.种植密度对籽瓜生长发育及产量的影响[J].新疆农业科学,2012,49(3):448-453.LIN Ming,LI Cheng-ye,PAN Jing-hai,et al.(2012).Effect of planting density on the growth and yield of seed-watermelon-SWBB-01[J]. Xinjiang Agricultural Sciences,49(3):448-453.(in Chinese)
[3]赵多勇,李应彪,翟金兰.籽瓜系列产品的开发现状与存在问题[J].农产食品科技,2008,2(1):45-47.ZHAO Duo-yong,LI Ying-biao,ZHAI Jin-lan,et al.(2013). Present Situation and Existing Problems in the Development of Watermelon Seed Products[J]. Agricultural Food Products Science and Technology,2(1):45-47.(in Chinese)
[4]羊杏平,刘广,侯喜林,等.利用RAPD和ISSR 2种分子标记鉴定西瓜杂交种的遗传纯度[J].江苏农业学报,2010,26(6):1 313-1 318.YANG Xing-ping,LIU Guang,HOU Xi-lin,et al.(2010).Evaluation of Genetic Purity of Watermelon Hybrid(Citrullus lanatus)Using PAPD and ISSR Molecular Markers[J]. Jiangsu Journal of Agricultural Sciences,26(6):1,313-1,318.(in Chinese)
[5]王吉明,李楠楠,尚建立,等.西瓜抗枯萎病种质资源的分子标记筛选研究[J].安徽农业科学,2015,43(32):200-202.WANG Ji-ming,LI Nan-nan,SHANG Jian-li,et al.(2015). Screening on Molecular Markers Lingked with Fusarium WiltResistance in Watermelon(Citrulls lanatus L.)Germplasam[J]. Journal of Anhui Agricultural Sciences,43(32):200-202.(in Chinese)
[6]汤谧,任俭,杜念华,等.分子标记技术在西瓜和甜瓜育种中的应用进展[J].贵州农业科学,2013,41(4):19-22.TANG Mi,REN Jian,DU Nian-hua,et al.(2013). Research Progress in Molecular Maker Technology Application in Watermelon and Melon Breeding[J]. Guizhou Agricultural Sciences,41(4):19-22.(in Chinese)
[7]张屹,刘建雄,梁志怀,等.不同西瓜品种枯萎病抗性鉴定方法比较[J].长江蔬菜,2015,(12):50-52.ZHANG Yi, LIU Jian-xiong, LIANG Zhi-huai, et al.(2015). Comparison of Resistance Identification Methods for Different Watermelon Cultivars[J]. Journal of Changjiang Vegetables,(12):50-52.(in Chinese)
[8]戴照义,王运强,刘志雄,等.西瓜种质资源的遗传多样性分析[J].中国瓜菜,2015,28(6):5-9.DAI Zhao-yi,WANG Yun-qiang,LIU Zhi-xiong,et al.(2015). Analysis on genetic diversity of watermelon germplasm resources[J]. China Cucurbits and Vegetables,28(6):5-9.(in Chinese)
[9]石磊,王萍,杨静,等.籽用西瓜种质资源SSR分析及初级核心种质库构建[J].西北植物学报,2016,36(6):1 125-1 134.SHI Lei,WANG Ping,YANG Jing,et al.(2016). Genetic Diversity Analysis and Construction of Seed Watermelon Core Germplasm Resources by SSR[J]. Acta Botanica Boreali-Occidentalia Sinica,36(6):1,125-1,134.(in Chinese)
[10]张楠,栾非时,高鹏. 4份野生瓜类材料抗病性及形态特征和SSR亲缘关系研究[J].园艺学报,2012,39(5):905-914.ZHANG Nan,LUAN Fei-shi,GAO Peng.(2012). The Study of Disease Resistant,Morphological Characteristics and Genetic Relationship Using SSR Markers in 4 Materials of Wild Cucurbit[J]. Acta Horticulturae Sinica,39(5):905-914.(in Chinese)
[11]Zietkiewicz,E.,Rafalski,A.,&Labuda,D.(1994). Genome fingerprinting by simple sequence repeat(ssr)-anchored polymerase chain reaction amplification. Genomics,20(2):176-183.
[12]李严,张春庆.西瓜杂交种遗传多态性的SRAP标记分析[J].园艺学报,2005,32(4):643-647.LI Yan,ZHANG Chun-qing.(2005). Studies on Genetic Diversity with a Molecular Marker SRAP of Watermelon Hybrids[J]. Acta Horticulturae Sinica,32(4):643-647.(in Chinese)
[13]李晓慧,田朝阳,王从彦. SRAP分子标记分析西瓜遗传多态性[J].生物技术,2007,17(3):23-26.LI Xiao-hui, TIAN Chao-yang, WANG Cong-yan.(2007). Genetic Polymorphism Analysis by Sequence-related Amplified Polymorphism Markers in Citrullus lanatus[J]. Biotechnology,17(3):23-26.(in Chinese)
[14]李朋飞,霍秀爱,程永强,等.基于SRAP的西瓜种质资源遗传多样性评价[J].中国农业科技导报,2013,15(2):89-96.LI Peng-fei,HUO Xiu-ai,CHENG Yong-qiang,et al.(2013). Assessment of Genetic Diversity in Watermelon Based on SRAP Analysis[J]. Journal of Agricultural Science and Technology,15(2):89-96.(in Chinese)
[15]易克,徐向利,卢向阳,等.利用SSR和ISSR标记技术构建西瓜分子遗传图谱[J].湖南农业大学学报(自然科学版),2003,29(4):333-337.YI Ke,XU Xiang Li,LU Xiang-yang,et al.(2003). Construction of Molecular Genetic Map of Watermelon by SSR and ISSR Technology[J]. Journal of Hunan Agricultural University(Natural Science),29(4):333-337.(in Chinese)
[16]羊杏平,刘广,侯喜林,等.利用RAPD和ISSR分子标记对西瓜作图亲本的多态性分析[J].江苏农业学报,2009,25(5):1 100-1 107.YANG Xing-ping,LIU Guang,HOU Xi-lin,et al.(2009).Polymorphism Analyses of Watermelon(Citrullus lanatus)Mapping Parents Using RAPD and ISSR Molecular Markers[J].Jiangsu Journal of Agricultural Science,25(5):1,100-1,107.(in Chinese)
[17]刘娟,廖康,赵世荣,等.利用ISSR分子标记构建新疆野杏核心种质资源[J].中国农业科学,2015,48(10):2 017-2 028.LIU Juan,LIAO Kang,ZHAO Shi-rong,et al.(2015). The Core Collection Construction of Xinjiang Wild Apricot Based on ISSR Molecular Markers[J]. Scientia Agricultura Sinica,48(10):2,017-2,028.(in Chinese)
[18]柳唐镜,于海彬,张棵,等.红籽瓜种质资源亲缘关系RAPD分析[J].中国农学通报,2010,26(22):264-270.LIU Tang-jing,YU Hai-bing,ZHANG Ke,et al.(2010).Genetic Relationships Analysis of Red-seed Edible Seed Watermelon Germplasm Resources by Using RAPD Markers[J]. Chinese Agricultural Science Bulletin,26(22):264-270.(in Chinese)
[19]刘海河,侯喜林,张彦萍.西瓜ISSR-PCR体系的正交优化研究[J].果树学报,2004,21(6):615-617.LIU Hai-he,HOU Xi-lin,ZHANF Yan-ping.(2004).Optimization of ISSR-PCR System in Watermelon[J]. Journal of Fruit Science,21(6):615-617.(in Chinese)
[20]黄歆贤. ISSR和SSR标记在大豆、油菜、西瓜种子真实性和纯度鉴定中的比较[D].杭州:浙江大学硕士学位论文,2011.HUANG Xin-xian.(2011). Comparison of ISSR and SSR markers for seed genuineness and variety purity identification in soybean,rape and watermelon[D]. Master Dissertation. Zhejiang University,Hangzhou.(in Chinese)
[21]杨生保,杨涛,李宁,等.番茄ILs果实性状的主成分分析与聚类分析[J].植物遗传资源学报,2015,16(4):907-913.YANG Sheng-bao,YANG Tao,LI Ning,et al.(2015). Principal Component and Cluster Analysis Based on Fruit Traits in ILs of Tomato[J]. Journal of Plant Genetic Resources,16(4):907-913.(in Chinese)
[22]杨生保,李宁,杨涛,等.基于SSR标记的制干辣椒品种资源遗传多样性分析[J].分子植物育种,2015,13(4):840-845.YANG Sheng-bao,LI Ning,YANG Tao,et al.(2015). Genetic Diversity of Dried-Orientation Pepper Cultivars Revealed by SSR Marker[J]. Molecular Plant Breeding,13(4):840-845.(in Chinese)
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